溶血磷脂質對牛腎上腺嗜鉻細胞乙醯膽鹼反應的影響

Abstract

溶血磷脂質(lysophospholipids)主要包含sphingosine-1-phosphate (S1P)和lysophosphatidic acid (LPA)，是在發炎與凝血反應中會大量產生的物質，主要由血液中的巨噬細胞與血小板產生。過去已經證實溶血磷脂質能調控細胞膜上離子通道活性。在這篇研究中，我們探討溶血磷脂質如何調控牛腎上腺嗜鉻細胞上乙醯膽鹼所造成的細胞內鈣離子濃度變化。結果顯示S1P前處理會降低嗜鉻細胞中由乙醯膽鹼造成的鈣離子反應；而LPA前處理亦會抑制乙醯膽鹼造成的內鈣濃度上升，且所需時間較短。乙醯膽鹼受器可分為兩大類:對muscarine反應的G蛋白耦合性受體和對nicotine反應的非專一性離子通道。細胞經溶血磷脂質處理後，會抑制nicotine刺激所引升的胞內鈣離子濃度反應，但對muscarine刺激所引升的胞內鈣離子濃度反應，則較無影響。以高鉀離子濃度的緩衝液刺激細胞，以檢驗電壓驅動式離子通道是否涉入溶血磷脂質引起的胞內鈣離子濃度反應時，結果顯示LPA會促進胞內鈣離子上升反應。從以上結果，我們發現S1P和LPA對乙醯膽鹼所造成的細胞內鈣離子反應有重要影響，表示S1P和LPA能調控腎上腺嗜鉻細胞的乙烯膽鹼受器活性。分析細胞內鈣離子濃度上升的原因，我們發現動作電位受溶血磷脂質作用而提前產生並且加速進行去極化與再極化；同時，動作電位受溶血磷脂質調控而降低振幅。這表示溶血磷脂質加速嗜鉻細胞的分泌速度並且減少分泌量來因應發炎或是受傷時的凝血反應。並且，LPA能在鈣離子通道受鎘離子阻斷時，大幅抑制動作電位振幅；但S1P無此現象。鈉離子通道電流決定動作電位去極化的過程。但溶血磷脂質不能影響鈉離子通道的電導、不活化現象以及回復現象。然而，以二次指數曲線密合鈉離子通道的回復曲線時，我們發現LPA處理過後的細胞有明顯不同的第二決定因子；這表示有一群鈉離子通道的回復現象受到LPA調控。只有進一步探討溶血磷脂質對鈣離子通道和鉀離子通道的調控才能使細胞調控乙烯膽鹼反應的過程更為明朗。

Lysophospholipids (LPLs), the chemicals secreted during inflammation affect ion channel activities. This study showed that sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), two important LPLs, modulated acetylcholine (ACh)-related responses in bovine chromaffin cells. S1P reduced ACh-induced intracellular calcium concentration ([Ca2+]i) responses. LPA had a similar effect as S1P pretreatment in inhibiting ACh-induced [Ca2+]i responses but with a faster timer course. ACh receptors (AChRs) could be classified into nicotinic- and muscarinic- types; the activation of both types increased the [Ca2+]i through different pathways. Chromaffin cells pretreated with LPLs showed that the [Ca2+]i responses were inhibited when cells were stimulated by 1,1-Dimethyl-4-phenylpiperazinium iodide, a stable nicotinic receptor agonist, but not muscarine. In the other hand, LPA enhanced high [K+]-induced [Ca2+]i responses, and this revealed some voltage-gated ion channels were affected. These suggested that LPLs played important roles in modulating chromaffin AChRs activities. To analyze the causes of escalation of intracellular calcium concentration, we found LPLs accelerated the depolarization and repolarization, and LPLs lowered the amplitude of action potential. This suggested LPLs accelerated and attenuated secretion to respond to inflammation and blood clotting. In addition, LPA pretreatment could make amplitude of action potential smaller when calcium channels were blocked while S1P pretreatment did not make the same reduction. Sodium current determined the depolarization of action potential, and LPLs did not change its conductance, inactivation and recovery. However, sodium currents recovery of LPA pretreatment showed a different second decisive factor with second-order exponential fitting. This means that a group of sodium channels was modulated by LPA. Only studying the calcium channels and potassium channels could further make clear the modulation of ACh responses.